The present invention relates to a gene conferring on a yeast, after transformation of the yeast cell, resistance to a herbicide-type growth inhibitor. More particularly, it concerns a plasmid wherein such a gene has been inserted in order to render a yeast cell transformed by the plasmid intrinsically resistant to the growth inhibitor. The invention also relates to a yeast made resistant to a herbicide-type growth inhibitor by transformation with such a plasmid. The present invention further concerns a process for the selective culture of yeast transformed by a plasmid containing a gene encoding resistance to a herbicide-type growth inhibitor.
It is known in the art to reprogram microorganisms genetically by transforming them with exogenous DNA under conditions that permit the DNA to replicate in the transformed cells, whereby the DNA is transmitted to the cells' progeny. The practical utility of these techniques is particularly evident when the exogenous DNA comprises at least one gene ensuring the expression by the transformed cell of a commercially valuable polypeptide, which could be either a hormone for medical use, like insulin, or an enzyme for industrial use, like amylase. Microorganisms thus transformed can be cultivated under suitable conditions so that they produce the polypeptide, which may then be recovered either from the microorganisms themselves or from the culture medium, depending on whether the polypeptide remains within the cultured cells or is excreted.
A laboratory method which is commonly used to transform a microorganism consists in using, as a vector for the exogenous gene of interest, an autoreplicating plasmid, i.e., a circular DNA molecule containing a sequence that makes it a replicon in the host cell. This plasmid also must contain at least one restriction site, i.e., a sequence specifically cleavable by a restriction enzyme.
If the plasmid once cleaved at a restriction site is the linked together with DNA fragments obtained either by excision from exogenous DNA or by in vitro synthesis, it is possible, using known techniques, to obtain so-called "chimeric" plasmids in which a fragment of exogenous DNA is inserted at the restriction site. After incubating a microorganism population in the presence of a DNA preparation containing such plasmids, it is possible to select from the population clones which have acquired, by transformation, one or more of the chimeric plasmids. The bank of clones thus obtained can then be screened in order to identify the one or several clones whose genome has been reprogramed by the DNA linked to the plasmid. Those clones are recognized either because they possess a new phenotypical feature which can easily be displayed by a culture on an indicator or selective medium, or because they respond positively to a test based on the use either of specific molecular probes, such as nucleic acids complementary to the gene sought, or of antibodies directed against the polypeptide encoded by the gene.
After identifying the clones sought, a fermentation process has to be developed which ensures an optimum production of the polypeptide of interest. In order to achieve this goal, two complementary approaches are usually applied: on the one hand, the chimeric vector is subjected to various in vitro manipulations in order to confer to the coding part of the corresponding gene a set of sequences best ensuring its expression; on the other hand, those culture conditions are selected which are best suited to the survival and proliferation of the transformed microorganisms, and to the exclusion of phenotypical revertants which might appear during fermentation. When such culture conditions have been defined, the fermentation process is genetically stable, and it can be used on an industrial scale.
It is known that the transformation of a microorganism according to the above methods most often produces an unstable transformant. The appearance of a growing proportion of phenotypically revertant cells in successive generations cultivated in a non-selective culture medium is a result of this unstability. An analysis above shows that such revertants have lost most or all of their plasmids (see K. Nagahari, J. Bacteriol. 136: 312, 1978). This situation results from the combination of two phenomena:
1. Spontaneous appearance within the culture of microorganisms partially or totally cured.
This curing can result from several causes:
statistical segregation of the plasmids during the cellular division; PA2 uncoupling of the replication of the plasmids and of the chromosomes; PA2 replication disadvantage of chimeric plasmids over endogenous plasmids (see C. P. Hollenberg, Curr. Top. Microbiol. Immunol. 96: 119, 1982).
2. Preferential proliferation of totally or partially cured microorganisms due to a selective disadvantage against the transformants (see K. Sakaguchi, in MOLECULAR BREEDING AND GENETICS OF APPLIED MICRO-ORGANISMS 1, Academic Press (ed. K. Sakaguchi & M. Okanishi 1980)).
This disadvantage is more pronounced when the expression of the genes contained in the plasmid is intense and requires a larger portion of cellular energy expenditures. This situation is paradoxical, since those wishing to develop a commercially viable process of industrial fermentation are primarily interested in maximizing expression of the cloned genes.
It is therefore necessary, in order to control the genetic stability of the population of microorganisms during fermentation, to exert an artificial selection pressure in favor of the transformants. This pressure can be exerted by adding to the culture medium a growth inhibitor towards which the vector-plasmid contains an extrinsic resistance marker. Most of the vectors used for the genetic manipulation of prokaryotic microorganisms confer on transformants a resistance to various antibiotics like ampicillin (inactivated by a .beta.-lactamase), neomycin (inactivated by a phosphorylase), and chloramphenicol (inactivated by an acetylase).
The selection pressure can also be exerted by carrying out the fermentation in an artificial culture medium devoid of an essential metabolite, the de novo synthesis of which is catalyzed by an enzyme encoded by a gene carried exclusively by the vector, the corresponding allele carried by the chromosome of the host having been inactivated by mutation. This approach is particularly applicable to yeast cells, which by transformation, using the most commonly used vectors can acquire the ability to grow in a medium free of leucine (pJDB207), of tryptophan (YRp7), or of uracil (pFL1).
Stability control generally constitutes no serious handicap for the setting up of small-scale industrial fermentation processes, such as those developed for the manufacture of medically useful products with very high added value, like hormones or vaccines. No major cost or supply problem arises with the first type of selection, while the use of an artificial culture medium with the second type of selection is acceptable from either a technical or an economical point of view; in either case, the amounts of product required remain relatively limited.
However, the need to resort to such practices considerably limits the prospects for setting up large scale industrial fermentation processes, such as those for the manufacture of large amounts of products with relatively low added value, such as certain enzymes with food processing use which are produced by transformed yeast. In this case, the use of artificial media devoid of an essential metabolite which the phenotypic revertants (as opposed to the transformants) are incapable of synthesizing, is particularly disadvantageous in light of the difficulty of preparing these media and their prohibitive prices.
On the other hand, the use of natural culture media which are relatively abundant and inexpensive, like lactoserum or molasses, has heretofore proven totally unworkable; such media are by nature non-selective because of their relatively high content of essential metabolites. In media of this type, the addition of significant amounts of antibiotics, such as chloramphenicol or a derivative of neomycin known as G418, an extrinsic resistance to which has been imparted to yeast by transformation (see C. P. Hollenberg, ICN-UCLA Symp. Mol. Cell. Biol. 15: 135, 1979; A. Jimenez & J. Davies, Nature 287: 869, 1980), would not only increase the fermentation costs significantly but would also entail serious supplying difficulties for those molecules for which the principal use was medical or veterinary. It is also known that yeast cells, like the cells of other eukaryots, are indifferent to most of the currently available antibiotics; it is deemed very unlikely, therefore, that other resistance markers to antibiotic-type inhibitors will be discovered for yeast and be profitably used to control stability in an efficient manner.